The major objective of the proposed research is to elucidate the structural changes in the adipocyte monosaccharide transport system that occur upon insulin activation. The approach to this problem will begin with the identification, purification, and characterization of the transport system in its unactivated state from rat fat cells. At the same time, experiments with isolated plasma membranes from insulin-treated and -untreated cells will be undertaken to determine whether insulin activation is due to an increase in the rates of already functioning transport systems or to an increase in the number of functional transport systems. The results from these two lines of investigation will determine the further course of the research to achieve the major objective. Three methods proposed for purification of the transport system from plasma membranes are: solubilization with detergents and separation of the protein-detergent complexes by conventional procedures; affinity chromatography of proteins in phospholipid vesicles, with a derivative of cytochalasin B, which is a high-affinity ligand for the transport system; immunoaffinity chromatography, with cross-reacting antibodies that have been prepared against the purified transporter from human erythrocytes. During purification the transport system will be assayed by its binding of cytochalasin B. A novel method to determine relative number of functioning transport systems in insulin-treated and -untreated cells is proposed: by dispersal of the transport systems into small vesicles so that there is one or none per vesicle, the intravesicular space available to D-glucose becomes a measure of relative number. Achievement of the major objective will increase our understanding, at the molecular level, of the effects of insulin deficiency in the disease, diabetes.